JP2000125871A - Isolation of catechol 2,3-oxygenation enzyme gene from environmental sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a primer capable of amplifying a fragment of catechol 2,3-oxygenation enzyme gene which is more various and close to the whole length. SOLUTION: The oligonucleotide contains a base sequence encoding a part or the whole of the amino acid sequence represented by Met-Asp-Phe-Met- Gly-Phe-Lys, Thr-Ile-Tyr-Phe-Phe-Asp-Pro or Leu-Asp-Arg-Met-Ala-Phe-Lys or a base sequence complementary to the base sequence. The isolation is carried out by performing the PCR by means of using this oligonucleotide as at least one side of PCR primers and the DNA of catechol 2,3-oxygenation enzyme productive bacteria as a template. By this process, it is possible to carry out the PCR amplification of catechol 2,3-oxygenation enzyme gene belonging to the different group difficult to amplify with the same primer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Many aromatic compounds are often environmental pollutants. On the other hand, microorganisms having enzymes that degrade aromatic compounds are present in the environment. The present invention relates to the field of environmental purification using microorganisms.

[0002]

2. Description of the Related Art In recent years, PCR has been used to rapidly obtain a specific gene sequence from a microorganism. However, the protein obtained from the gene sequence obtained by the PCR method does not always retain the function, and in order to obtain the gene of the enzyme having the function, it was necessary to search for bacteria and clone the gene. Methods for obtaining functional genes by performing two-stage gene amplification have been developed. By using this method, the target enzyme gene can be isolated without going through complicated processes such as isolation of bacteria and gene screening.

[0003]

In the method using the primers designed so far, the obtained catechol 2,3-
All oxygenase genes belong to the group classified as I.2.A, and have very similar DNA sequences. The gene region that can be amplified with this primer is 451 bp,
21 bp). From these facts, it was necessary to design new primers in order to obtain a more diverse catechol 2,3-oxygenase gene. The present invention has been made in view of such a demand, and an object of the present invention is to provide a primer capable of amplifying a more diverse and nearly full-length catechol 2,3-oxygenase gene fragment.

[0004]

Means for Solving the Problems As a result of repeated studies to achieve the above object, the present inventors have conserved the catechol 2,3-oxygenase I.2.A and I.2.B subfamilies. The present inventors have found that there is an amino acid sequence having a high possibility, and have completed the present invention based on this finding. That is, the first aspect of the present invention is to PCR-amplify the gene sequence of the catechol 2,3-oxygenase gene from an environmental sample in a longer range than before using the nucleotide sequence of the catechol 2,3-oxygenase gene, Is to be isolated. Here, the base sequence is P. putida
Met-Asp-Phe-Met of catechol 2,3-oxygenase of MT2 strain
-Gly-Phe-Lys amino acid sequence and Thr-Ile-Tyr-Ph
A partial base sequence of a 642 bp catechol 2,3-oxygenase gene encoding an amino acid sequence flanked by amino acid sequences represented by e-Phe-Asp-Pro can be used.

[0005] These primers are provided with an additional sequence for preparing a functional gene. That is, each primer is a catechol 2,3-oxygenase Ala-Asp-Glu-Pro-Gly-Met-Asp-Phe-Met-Gly-Ph of the P. putida MT2 strain.
Amino acid sequence represented by e-Lys and Thr-Ile-Tyr-Phe-Phe-A
This corresponds to the amino acid sequence represented by sp-Pro-Ser-Gly-Asn-Arg-Asn-Glu-Val.

[0006] A second aspect of the present invention is to use the base sequence of the catechol 2,3-oxygenase gene from environmental samples using the base sequence of the catechol 2,3-oxygenase gene. It is to amplify the gene sequence classified as B at the same time. Here, as the base sequence, the catechol 2,3- oxygenase Leu-Asp-Arg-Met of S. yanoikuyae B1 strain is used .
-Ala-Phe-Lys amino acid sequence and T
hr-Ile-Tyr-Phe-Phe-Asp-Pr
A partial base sequence of a catechol 2,3-oxygenase gene encoding an amino acid sequence flanked by amino acid sequences represented by o can be used.

[0007] These primers are provided with an additional sequence for preparing a functional gene. That is, each primer is a catechol 2,3-oxygenase Glu-Ala-Asp-Ser-Ala-Gly-Leu-Asp-Arg-Met-Al of P. putida MT2 strain.
Amino acid sequence represented by a-Phe-Lys and Ile-Tyr-Phe-Phe-A
This corresponds to the amino acid sequence represented by sp-Pro-Ser-Gly-Asn-Arg-Asn-Glu.

Hereinafter, the present invention will be described in detail. The present invention details PCR primers used to obtain a functional gene by a continuous gene amplification reaction. DN containing a specific nucleotide sequence on the catechol 2,3-oxygenase gene
By amplifying the A fragment by the PCR method and using the three PCR products in a continuous gene amplification reaction, a functional gene that cannot be obtained by a single PCR can be obtained.

[0009] Primers used for PCR include Ala-Asp
-Glu-Pro-Gly-Met-Asp-Phe-Met-Gly-Phe-Lys forward primer 1 or Ala-Asp-Ser-Ala-Gly-Leu-Asp containing the nucleotide sequence encoding the amino acid sequence represented by -Arg-M
et-Ala-Phe-Lys forward primer 2 containing a nucleotide sequence encoding the amino acid sequence represented by, Thr-Ile-Tyr-P
reverse primer 1 or Ile-Tyr-Phe-Phe-Asp-Pro- containing the nucleotide sequence encoding the amino acid sequence represented by he-Phe-Asp-Pro-Ser-Gly-Asn-Arg-Asn-Glu-Val Ser-Gly-A
Two types of primers, a reverse primer 2 containing a nucleotide sequence encoding the amino acid sequence represented by sn-Arg-Asn-Glu, are used. The template DNA binding site of each primer of the forward primer 1 and the reverse primer 1 is P.putida MT-
Two strains catechol 2.3-oxygenase gene sequence (PPXYLE [G
enBank]), pos. 196-212 and 757-777, respectively. The template DNA binding site of each primer of the forward primer 2 and the reverse primer 2 is S. yanoikuyae B1 strain catechol 2.3-oxygenase gene sequence (U23375 [GenBan
k]) correspond to pos. 196-212 and 760-776, respectively. By using these primers, catechol 2,3-
Amino acid sequence represented by Met-Asp-Phe-Met-Gly-Phe-Lys or Asp-Arg-Met-Ala-Phe-Lys on oxygenase and T
hr-Ile-Tyr-Phe-Phe-Asp-Pro or Ile-Tyr-Phe-Phe-
A DNA fragment containing a partial base sequence of catechol 2,3-oxygenase encoding an amino acid sequence flanked by amino acid sequences represented by Asp-Pro can be amplified. The forward primer and the reverse primer are designed based on a seven amino acid sequence as shown in FIGS. 1, 2, 3 and 4. Of these amino acids,
Only four out of six codons for arginine were used, and for the other amino acids, primers were created to cover all of the codons, resulting in 256, 32, and 7 codons.
68 types and 192 types are mixed.

[0010] These primers are added with a DNA sequence necessary for preparing a functional gene. The additional sequence of each primer of the forward primer 1 and the reverse primer 1 is Ala-Asp-Glu-Pro-Gly and Ser-Gly-Asn-Arg described above.
-Asn-Glu-Val, which corresponds to P. putida strain catechol 2.3-oxygenase gene sequence pos. 178-192, 783-
797 respectively. Also, forward primer
2, additional sequence of each primer of reverse primer 2,
Ala-Asp-Ser-Ala-Gly-Leu and Ser-Gly-Asn-Arg-A above
This sequence corresponds to sn-Glu, and this sequence corresponds to S. yanoikuyae B1 strain catechol 2.3-oxygenase gene sequence pos. 178-195, 777-
The amplified DNA sequence corresponding to 794 is a partial sequence of catechol 2,3-oxygenase, and the protein produced from this PCR fragment has no function. For this purpose, a functional gene is constructed by connecting a known catechol 2,3-oxygenase sequence to a gene region that cannot be amplified by a single reaction. Catechol 2,3- obtained by the present invention
Oxygenases belong to the subfamilies classified as I.2.A and I.2.B, and catechol belonging to the subfamilies I.1, I.2.C to D, and I.3 to 5. It is unlikely that a 2,3-oxygenase gene will be obtained.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The oligonucleotide of the present invention comprises
An amino acid sequence represented by Met-Asp-Phe-Met-Gly-Phe-Lys, an amino acid sequence represented by Thr-Ile-Tyr-Phe-Phe-Asp-Pro, Leu-Asp-Arg-Met-Ala- Includes a base sequence encoding part or all of the amino acid sequence represented by Phe-Lys or a base sequence complementary thereto. Met-Asp-Phe-Met-Gly-Phe-Ly
The amino acid sequence represented by s is the partial amino acid sequence of catechol 2,3-oxygenase of P. putida MT2 strain, Leu-As
The amino acid sequence represented by p-Arg-Met-Ala-Phe-Lys is S.
2 is a partial amino acid sequence of a catechol 2,3-oxygenase of yanoikuyae B1 strain. Also, Thr-Ile-Tyr-Phe-Phe-Asp-
Pro is the amino acid sequence contained in the catechol 2,3- oxygenase of both the P. putida MT2 strain and the S. yanoikuyae B1 strain.

In the method for isolating the catechol 2,3-oxygenase gene of the present invention, the above-mentioned oligonucleotide is used as at least one PCR primer, and the DNA of catechol 2,3-oxygenase-producing bacteria is used as a template to perform PCR. It is characterized by performing. Preferred oligonucleotides used as the forward primer include (1) Met-Asp-Phe-Met-Gly-Ph
Oligonucleotides represented by the base sequence encoding e-Lys, and oligonucleotides represented by the base sequence encoding (2) Leu-Asp-Arg-Met-Ala-Phe-Lys can be exemplified. In addition, additional sequences were added to these oligonucleotides (3) Ala-Asp-Glu-Pro-Gly-Met-Asp-Phe-Me
an oligonucleotide represented by a nucleotide sequence encoding t-Gly-Phe-Lys, (4) Ala-Asp-Glu-Pro-Gly-Leu-Asp-
An oligonucleotide represented by a base sequence encoding Arg-Met-Ala-Phe-Lys can also be exemplified.

Preferred oligonucleotides used as the reverse primer include (5) Thr-Ile-Tyr-Phe-
Oligonucleotide represented by a nucleotide sequence complementary to the nucleotide sequence encoding Phe-Asp-Pro, (6) Ile-Tyr-Phe-
An oligonucleotide represented by a nucleotide sequence complementary to a nucleotide sequence encoding Phe-Asp-Pro can be exemplified.
In addition, additional sequences were added to these oligonucleotides.
(7) Thr-Ile-Tyr-Phe-Phe-Asp-Pro-Ser-Gly-Asn-Arg-As
an oligonucleotide represented by a nucleotide sequence complementary to the nucleotide sequence encoding n-Glu-Val, (8) Ile-Tyr-Phe-Ph
Oligonucleotides represented by a nucleotide sequence complementary to the nucleotide sequence encoding e-Asp-Pro-Ser-Gly-Asn-Arg-Asn-Glu-Val can also be exemplified.

Preferred combinations of the forward primer and the reverse primer include (1) and (5), and (2)
(6), (3) and (7), and (4) and (8) can be exemplified, but are not limited thereto. (1) and (5) or (3) and (7)
When PCR is performed using the combination of the primers described above, the gene sequence of the catechol 2,3-oxygenase gene can be amplified in a longer range (specifically, 642 bp) than before.
Combination of primers (2) and (6) or (4) and (8)
When PCR is performed, not only can the gene sequence be amplified in a longer range than before, but the gene sequence that is classified as I.2.A and I.2.B of the catechol 2,3-oxygenase gene can be It can be amplified simultaneously.

[0015]

[Example] Example: Application to a method for obtaining a functional gene by continuous gene amplification reaction Using the forward primer 2 and the reverse primer 2 shown in Figs. 2 and 4, P. putida MT2 strain and dibenzothiophene degradation From a mixed culture of bacteria and phenanthrene-degrading bacteria
R amplification was attempted. As a result, P. belonging to the I.2.A family .
The amplification of the catechol 2,3-oxygenase gene of putida MT2 strain was confirmed. From the mixed culture system of divanzothiophene and phenanthrene-degrading bacteria, three different catechol 2,
The gene sequence of 3-oxygenase was obtained. Analysis of these gene sequences revealed that they all belonged to the catechol 2,3-oxygenase I.2.B subfamily.

[0016]

According to the present invention, catechol 2, which belongs to different groups and is difficult to amplify with the same primer,
PCR amplification of 3-oxygenase gene became possible.

[Brief description of the drawings]

FIG. 1 shows a forward primer 1 used in the present invention.

FIG. 2 shows forward primer 2 used in the present invention.

FIG. 3 shows a reverse primer 1 used in the present invention.

FIG. 4 shows reverse primer 2 used in the present invention.

FIG. 5 shows the amino acid sequence of the catechol 2,3-oxygenase isolated by the present inventors.

 ────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Shigeaki Harayama 75-1 Hirata 3rd Land 75, Kamaishi City, Iwate Prefecture F-term in Kamaishi Research Institute, Marine Biotechnology Institute 4B024 AA17 AA20 BA08 CA09 HA12 HA20 4B063 QA05 QA13 QA18 QQ44 QR32 QR39 QR55 QR62 QS25 QS34

Claims (2)

[Claims] 1. An amino acid sequence represented by Met-Asp-Phe-Met-Gly-Phe-Lys, an amino acid sequence represented by Thr-Ile-Tyr-Phe-Phe-Asp-Pro, Leu-Asp-Arg -An oligonucleotide comprising a base sequence encoding a part or all of the amino acid sequence represented by Met-Ala-Phe-Lys or a base sequence complementary thereto. 2. The method according to claim 1, wherein PCR is carried out using the oligonucleotide of claim 1 as at least one PCR primer and DNA of a catechol 2,3-oxygenase producing bacterium as a template. A method for isolating enzyme genes.